Human beings are exposed to a wide variety of substances and processes that are known to be, or are suspected of being, mutagenic. Such exposure may result from a wide variety of sources, including toxic wastes, technologically innovative products, and byproducts of common substances. To help safeguard society from mutagenic agents, it is necessary to measure the ability of an agent to create or promote alterations in the genetic composition and reproduction of cells and animals. Mutagenicity assays allow for such measurements in laboratories.
Mutagenicity assays typically are conducted by exposing a cell culture to a substance or process that is suspected of being mutagenic. After the exposure of the suspected mutagen is terminated, the culture normally is allowed to grow for a period of time necessary to allow the mutant phenotype to be expressed, called a "phenotypic expression period;" see, for example, U.S. Pat. No. 4,066,510 (Thilly, 1978). The mutant frequency of the exposed culture is then compared with a control culture to determine whether the mutagen exposure induced a higher frequency of genetic change.
Humans suffer from three distinct forms of genetic change: (a) having an abnormal number of chromosomes, (b) having an abnormal structure of one or more chromosomes, (c) having an abnormal sequence in the DNA that constitutes the genetic material of the chromosomes[1].
Abnormalities of chromosome number and structure can often be detected by direct microscopic observation of condensed chromosomes of cells in mitosis. Abnormalities in DNA sequence usually are not microscopically detectable, and require indirect means of detection known generally as "gene locus mutation assays."
The terms "mutation assay" or "mutagenicity assay" are used interchangeably herein to include assays which detect any type of genetic change, such as change in chromosomal number or structure, or change in gene locus.
Genes which encode the information for making an enzyme or protein catalyst are especially useful in mutation assays. Cells carrying only one copy of such a gene can lose the ability to make the enzyme through a single mutation in that gene. Genes located on the sex chromosomes (x and y) are present in only one active copy per cell. Genes located on autosomes are normally present in two copies per cell. It is sometimes possible to select a heterozygote, a cell with only one functional copy of an autosomal gene [2].
The presence or absence of an enzyme within cells may be determined by adding a "selective agent" to the nutrient solution in which the culture is growing so that only cells without the enzyme can grow. The term "selective agent" has been extensively described and is understood by those skilled in the art. Many selective agents are structurally similar to non-toxic molecules that are metabolized by the cell. For example, 6-thioguanine (6-TG) is a toxic analog of guanine; the structure of each is shown below. ##STR1##
A toxic analog such as 6-TG wiII be utilized by a cell only if a certain enzyme is present in the cell. Cells which lack that particular enzyme will survive the exposure to the toxic analog. Cells which possess the enzyme in normal amounts will be killed by the toxic analog. In this way, cells which lack certain enzymes can be selected.
Mutation assays have been performed using a number of selective agents of this nature, including the following: 6-TG, which kills cells that contain hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) [3]; 8-azaguanine, which kills cells that contain HGPRT [4]; and trifluorodeoxythymidine, which kills cells that contain thymidine kinase [5].
In a typical mutation assay, replicate cell cultures are exposed to different concentrations of a test chemical (a suspected or known mutagen). A control culture is handled identically in all aspects except it is not exposed to the test chemical. After recovery from the mutagen exposure and phenotypic expression of any newly induced mutations, the relative survival in the presence of the selective agent for each cell culture is determined. This relative survival frequency (the number of cells in the cell culture which grow to form colonies in the presence of the selective agent, divided by the number of cells capable of forming colonies)is called the mutant fraction. A significant increase in mutant fraction indicates that the test chemical was mutagenic.
Many such assays involve the cells of bacteria and other lower organisms, and mice and other small mammals. However, the results of such experiments cannot always be extrapolated to determine the mutagenicity of a substance or process when exposed to human cells. Therefore, a number of mutagenicity assays have been developed which incorporate cells of human origin, grown in vitro [6].
Various types of cells in humans contain certain enzymes that metabolize various substances called "xenobiotics." Xenobiotics are foreign substances which do not normally exist within humans and are frequently toxic, and therefore must be excreted. When human cells are isolated from an individual and grown in long term cell culture, the cells tend to lose the ability to metabolize xenobiotics. If it is desired to conduct a mutagenicity assay that involves one or more xenobiotic substances, then it frequently is necessary to add to the cell cultures an exogenous xenobiotic metabolizing system. These systems commonly include tissues homogenate or whole cells isolated from an animal. The enzymes which carry out xenobiotic metabolism are exogenous, i.e., they act outside of the human cells being assayed. By contrast, "endogenous" enzymes are created, and normally act, within the cells being assayed. The most common source of exogenous xenobiotic-metabolizing enzymes is rodent (mouse or rat) liver. These enzymes are usually prepared by removing the liver from the animal, homogenizing the liver tissue, and centrifuging the homogenate to remove the larger particles such as outer cell membranes, nuclei and mitochondria. The resulting supernatant is referred to as post-mitochondrial supernatant (PMS) [7].
Many types of normal tissues,and PMS preparations from such tissues, are capable of xenobiotic metabolism. Such metabolism tends to occur at relatively high rates in certain types of cells, such as liver cells and bronchial cells. The normal function of this metabolism is to convert non-polar (lipophilic) xenobiotic compounds to more polar, water-soluble forms which can be more easily excreted by the body. Occasionally during this process, chemically reactive metabolites such as epoxides are produced. Some of the reactive metabolites can cause mutations. The level of several xenobiotic metabolizing activities can be increased (or induced) by treatment with certain xenobiotics, such as benzo(.alpha.)pyrene and beta-naphthoflavone.
Several types of enzymatic activities are capable of converting various xenobiotic compounds into potentially mutagenic metabolites. Several activities which are of particular interest herein are designated as oxygenase, peroxidase, oxidase and hydroxylase activities. Those terms are sometimes used improperly and inconsistently in scientific articles, and various reference works [8]should be referred to for exact definitions used by those skilled in the art. In general, these enzymes catalyze the addition of oxygen by adding oxygen atoms or oxygen-containing moieties, or by withdrawing electrons. For convenience, the term "oxidative" is used herein to include oxygenase, peroxidase, oxidase, and hydroxylase activity.
If a hydroxylase enzyme acts upon an aryl hydrocarbon substrate molecule, such activity may be referred to as "aryl hydrocarbon hydroxylase" (AHH) activity. AHH activity can produce phenols and epoxides from polynuclear compounds, as shown by the following example reactions: ##STR2##
In the reactions shown above, double bonds are assigned to specific locations so that the bonding reactions are compatible. However, in most aromatic compounds, the electrons are in resonant configurations.
An enzyme which can act upon a variety of substrate molecules is commonly called a "mixed function" enzyme.
In their reduced state and in the presence of carbon monoxide, certain mixed function oxidative enzymes absorb light with a wavelength maximum around 450 nanometers (nm). Because of this characteristic, such enzymes are often referred to as "cytochrome P-450" enzymes. These enzymes are present in rodent liver PMS, and are capable of mixed function oxidative activity. Cytochrome P-450 enzymes include several distinct isozymes.
The use of PMS preparation, or other sources of exogenous enzymes, in a mutation assay involving human or other cells, may lead to several problems. Such problems include:
1. Enzymes from different species of animals are likely to differ in their chemical makeup and metabolic processes. This leads to uncontrolled variations and uncertainties in the biochemical reaction.
2. Exogenous enzymes usually do not enter the cells; instead, they normally perform their specialized functions on molecules that are outside of cells. This differs from the normal function of these enzymes, which normally act within cells.
3. Exogenous enzymes are likely to create different metabolities compared to endogenous enzymes. For example, an oxygen atom might be bonded to any of several different carbon atoms within an aromatic molecule to create numerous different types of epoxides or phenols. The epoxides and phenols that result from varying enzymes may vary structurally, and may have differing characteristics and biochemical functions.
4. The preparation and addition of exogenous enzymes requires delay and expense. Also, the addition of exogenous enzymes must be carefully controlled in regard to numerous parameters, such as pH and concentration, and steps must be taken to ensure sterility.
5. Homogenate preparations typically contain numerous enzymes and other biochemicals in addition to the specific enzymes desired. Such impurities can lead to reactions that differ from or interfere with the desired reaction. In addition, such impurities may exert toxic effects.
Certain cells are available which are known to possess relatively high levels of endogenous oxygenase activity [9]. However, such cells tend to suffer from various problems when used in mutagenicity assays. Those problems include:
1. Many of the cells are not of human origin. 2. Many of the cells are not diploid, i.e., they often possess an abnormal number (more or less than two copies) of some chromosomes. Such cells tend to be genetically unstable, which interferes with accurate analysis of mutagenicity assay results. 3. Such cells tend to grow poorly in culture, for reasons which include slow growth rates, poor colony forming efficiency, and limitations on the number of generations that can be grown in culture. 4. Such cells may contain various types of contamination, such as mycoplasma, which interfere with accurate measurement of mutation. 5. Many such cells lines are non-homogeneous, i.e., they contain subpopulations with characteristics that differ from the remainder of the cells. Such subpopulations may contain lower levels of oxidative activity, which may lead to selective advantages during mutagenicity assays that involve relatively toxic concentrations of suspected mutagens. Such subpopulations interfere with accurate analysis of mutation. 6. Most cells which contain endogenous oxidative activity, such as certain types of cells from the liver or respiratory tract, are anchorage-dependent cells. Anchorage-dependent cells proliferate poorly unless they are allowed to contact a solid surface, such as a microcarrier bead or the wall of a culture flask or roller bottle. This characteristic requires special culturing techniques which increase the time, effort, and expense required to grow such cells. By contrast, cells which are not anchorage-dependent, such as lymphoblasts and other blood cells, can grow in suspension cultures, which are normally stirred to prevent the cells from settling to the bottom of the culture medium. Such cells can usually be grown with less effort and expense than anchorage-dependent cells. In addition, it is easier to obtain samples of blood cells than to obtain samples of anchorage-dependent cells.